Immunity enhancing lactic acid bacteria

ABSTRACT

Novel bacteria  Lactobacillus rhamnosus  HN001 and HN 067,  Lactobacillus acidophilus  HN017, and  Bifidobacterium lactis  HN019 are claimed. Each strain provides immune enhancing effects when ingested.

TECHNICAL FIELD

[0001] This invention relates to novel strains of lactic acid bacteriaand their use in enhancing immunity.

BACKGROUND ART

[0002] The consumption of products containing lactic acid bacteria (LAB)is associated with a range of health benefits including enhancement ofimmunity. There are thousands of strains of lactic acid bacteria butonly some strains exhibit health-promoting properties. The ability ofthese bacteria to tolerate acids and bile salts, adhere to mucosalepithelial cells, and to survive passage through the gastrointestinaltract is considered an important criterion for selection ofhealth-promoting strains. Only a few strains of lactic acid bacteriawith proven health benefits have been identified to date.

[0003] Strains of LAB showing good adhesion to the cells of the mucosalepithelium of the small intestine thereby lending themselves totherapeutic applications are known from New Zealand Patent 248057. Themicro-organisms described in this patent enhance both natural immunity(phagocyte function) and acquired immunity (antibody responses andlymphocyte proliferation responses).

[0004] It is desirable to have other LAB bacteria that enhance a broadspectrum of immune responses including phagocyte function.

[0005] It is an object of this invention to go some way towardsachieving these desiderata or at least to offer the public a usefulchoice of immune enhancing lactic acid bacteria.

DISCLOSURE OF THE INVENTION

[0006] Accordingly, in one aspect the invention may be said broadly toconsist of a biologically pure culture of Lactobacillus rhamnosus HN001,AGAL deposit number NM97/09514 dated Aug. 18, 1997.

[0007] In another aspect the invention may be said broadly to consist ofa biologically pure culture of Lactobacillus rhamnosus HN067, AGALdeposit number NM97/01925 dated Feb. 17, 1998.

[0008] In another aspect the invention may be said broadly to consist ofa composition of a biologically pure culture of any one of Lactobacillusacidophilus HN017, AGAL deposit number NM97/09515 dated Aug. 18, 1997,Lactobacillus rhamnosus HN001, Lactobacillus rhamnosus HN067 orBifidobacterium lactis HN019, AGAL deposit number NM97/09513 dated Aug.18, 1997 in an immunostimulating concentration, with a physiologicallyacceptable excipient or diluent.

[0009] In one embodiment said composition contains any two or more ofsaid strains.

[0010] Preferably said physiologically acceptable excipient or diluentis a food.

[0011] Preferably said food is any one of cultured milk, yoghurt,cheese, milk drink or milk powder.

[0012] Alternatively said composition is a pharmaceutical compositionand said excipient or diluent is pharmacologically acceptable excipientor diluent.

[0013] Immunity enhancing, physiologically acceptable, biologically purestrains of homologues or mutants of any one of the strains:

[0014]Lactobacillus acidophilus HN017,

[0015]Lactobacillus rhamnosus HN001,

[0016]Bifidobacterium lactis HN019, or

[0017]Lactobacillus rhamnosus HN067.

[0018] In another embodiment the invention may be said broadly toconsist of a method of enhancing natural and acquired immunity whichcomprises administering to a mammal any one of the above biologicallypure cultures at an immunostimulating dosage rate.

[0019] In another embodiment substantially biologically pure cultures oftwo or three of the above-defined strains are present.

[0020] Preferably said culture is administered in the form of acomposition with a physiologically acceptable excipient or diluent.

[0021] Preferably said physiologically acceptable excipient or diluentis a food.

[0022] Preferably said food is cultured milk, yoghurt, cheese, milkdrink or milk powder.

[0023] This invention may also be said broadly to consist in the parts,elements and features referred to or indicated in the specification ofthe application, individually or collectively, and any or allcombinations of any two or more of said parts, elements or features, andwhere specific integers are mentioned herein which have knownequivalents in the art to which this invention relates, such knownequivalents are deemed to be incorporated herein as if individually setforth.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 shows the effect of supplementation of mice with productfermented with L. rhamnosus HN001 or unfermented product containing L.rhamnosus HN001 on phagocyte activity of peripheral blood leukocytes asdescribed in example 5. BALB/c nice were fed on milk based dietscontaining 10⁹ cfu (per day) L. rhamnosus HN001 in either fermented orunfermented product for 14 days. Phagocytic activity of peripheral bloodleukocytes was determined using flow cytometry and fluorosceinisothiocyanate-labelled Escherichia coli. Values are mean±standarderror. Significant differences (ANOVA, the SAS program) from thecontrol: **P<0.0001.

[0025]FIG. 2 shows the effect of supplementation of mice with live L.rhamnosus HN001 or heat killed L. rhamnosus HN001 on phagocytic activityof peripheral blood leukocytes as described in example 7. BALB/c micewere fed on milk based diets and orally administered 10⁹ cfu (per day)of either live or heat killed L. rhamnosus HN001 for 14 days. Phagocyticactivity of peripheral blood leukocytes and peritoneal macrophages weredetermined using flow cytometry and fluoroscein isothiocyanate—labelledEscherichia coli. Values are mean±standard error. Significantdifferences (ANOVA, the SAS program) from the control, **P<0.0001.

[0026]FIG. 3 shows the effect of supplementation of mice with L.rhamnosus HN001 or B. lactis HN019 on bacteria translocation in micechallenged with S. typhimurium as described in example 8. Unsupplementedand B. lactis HN019, or L. rhamnosus HN001 supplemented BALB/c mice wereorally challenged with S. typhimurium following continuous dailysupplementation. Six days after challenge mice were humanely killed andtheir livers and spleens were harvested for monitoring bacterialtranslocation. Tissue suspensions from the harvested organs were thencultured on MacConkey agar plates for 24-48 hr prior to enumeration.Values are mean±standard error. Significant differences (ANOVA, the SASprogram) from the control: *P<0.05.

[0027]FIG. 4 shows the effect of supplementation of mice with L.rhamnosus HN001 or B. lactis HN019 on the phagocytic activity ofperipheral blood leukocytes from mice challenged with S. typhimurium asdescribed in example 8. Unsupplemented and B. lactis HN019, or L.rhamnosus HN001 supplemented BALB/c mice were orally challenged with S.typhimurium following continuous daily supplementation. Phagocyticactivity of peripheral blood leukocytes was determined six days afterchallenge using flow cytometry and fluoroscein isothiocyanate-labelledEscherichia coli. Values are mean±standard error. Values (mean±standarderror) with different superscripts are significantly different (ANOVA,the SAS program): P<0.01.

[0028]FIG. 5 shows the effect of supplementation of mice with L.rhamnosus HN001 or B. lactis HN019 on the proliferative responses ofspleen lymphocytes from mice challenged with S. typhimurium as describedin example 8. Unsupplemented and B. lactis HN019, or L. rhamnosus HN001supplemented BALB/c mice were orally challenged with S. typhimuriumfollowing continuous daily supplementation. Six days after challenge theproliferative responses of spleen lymphocytes were measuredcolourimetrically following the incorporation of 5-bromo-2′-deoxyuridinefor the final 16 hrs of the 96 hr incubation. Values (mean±standarderror) with different superscripts are significantly different (ANOVA,the SAS program): P<0.01).

MODES OF CARRYING OUT THE INVENTION

[0029] Freeze dried cultures of the four bacterial strains have beendeposited at the Australian Government Analytical Laboratories (AGAL),The New South Wales Regional Laboratory, 1 Suakin Street, Pymble, NSW2073, Australia. Details of the deposits are: Strain Number Date L.acidophilus HN017 NM97/09515 August 18 1997 L. rhamnosus HN001NM97/09514 August 18 1997 B. lactis HN019 NM97/09513 August 18 1997 L.rhamnosus HN067 NM97/01925 February 11 1998

[0030] The four strains identified above have been found to enhance abroad spectrum of immune responses including both natural and acquiredimmune responses.

EXAMPLE 1 Morphology and General Properties

[0031] RAPD analysis, 16S rRNA sequencing and SDS-PAGE analyses wereused to confirm taxonomical characterisation of strains. It was alsofound that L. acidophilus HN017 was genetically different from L.acidophilus (LC1) of New Zealand Patent No.248057.

[0032] RAPD analysis, 16S rRNA sequencing and SDS-PAGE analyses wereused to confirm taxonomical characterisation of L. rhamnosus HN067;species-specific primers used for characterisation of L. rhamnosus HN067at molecular level included Pr I (forward) 5-CAGACTGAAAGTCTGACGG-3 andPha II (reverse) 5-GCGATGCGAATTTCTATTATT-3.

[0033] The morphology and sugar fermentation properties of this strainare detailed in Tables 1 and2. TABLE 1 Morphology and othercharacteristics L. acidophilus L. rhamnosus B. lactis L. rhamnosus HN017HN001 HN019 HN067 Short to medium Short to medium Microaerophilic toShort to medium rods with rounded rods with square anaerobic rods withrods with square ends, generally ends in chains, characteristic shapesends in chains, occurring singly generally 0.7 × such as middlegenerally 0.7 × 1.1 × or in pairs or short 1.1 × 2.0-4.0 μm, enlargedcells, ‘V’ or 2.0 to 4.0 μm, chains, when when grown in palisadearrangement when grown in grown in MRS MRS broth. of cells when grownMRS broth. broth. Gram positive, on TPY agar slabs. Gram positive, Grampositive, non-mobile, non- In MR5 broth with catalase negative,non-spore spore forming, 0.05% cysteine non-mobile, non forming,catalase catalase negative hydrochloride, they spore-forming, negativefacultative form middle-enlarged facultative facultatively anaerobicrods cells and club shaped anaerobic rods anaerobic rods with optimum(spatulated with optimum with optimum growth extremities) cells, growthgrowth temperature of Gram positive, non- temperature of temperature of37 ± 1° C. and motile and non-spore 37 ± 1° C. and 37 ± 1° C. andoptimum pH of forming, catalase optimum pH of 6.0 optimum pH of 6.0-6.5.These negative rods with to 6.5. These are 6.0-6.5. These arefacultatively optimum growth facultatively are obligatelyheterofermentative temperature of heterofermentative homofermentativebacteria and no 37 ± 1° C. and optimum bacteria and no bacteria and nogas produced from pH of 6.0-7.0. gas produced from gas is producedglucose. Fructose-6-phosphate glucose. from glucose. phospho-ketolasepositive.

[0034] TABLE 2 Carbohydrate fermentation pattern of selectedLactobacillus and Bifidobacterium strains S1. No. Name of the bacteriumScore* 1 L. acidophilus HN 017 5755546 2 L. rhamnosus HN001 5757177 3 B.lactis HN019 1051622 4 L. rhamnosus HN067 5757175

EXAMPLE 2 Adhesion to Intestinal Cells

[0035] The ability of probiotic strains to adhere to human intestinalepithelial cells (HT-29 and CaCo-2) was assessed in vitro usingdifferentiated cell-lines. Monolayers of HT-29 and CaCo-2 cells weregrown on cover slips and placed in multi-well dishes. 10⁸ cfu/ml of LABin 1 ml of spent culture supenatant was then added to cell layers alongwith 1 ml of DMEM medium and incubated for 1 hr at 37° C. in 10% CO₂-90%air. Monolayers were washed 4 times with PBS, fixed in methanol, Gramstrained and the number of bacteria adhering to epithelial cellsdetermined microscopically. On average, 20 fields were counted and theresults are summarised in Table 3. TABLE 3 Adherence to HT-29 and CaCo-2cell lines* STRAIN HT-29 CaCo-2 L. acidophilus HN 017  98 ± 17 171 ± 16L. rhamnosus HN 001 161 ± 18 218 ± 35 B. lactis HN 019 188 ± 27 194 ± 25

EXAMPLE 3 Enhancement of Natural and Acquired Immunity

[0036] The immunoenhancing effects of the three strains L. rhamnosusHN001, L. acidophilus HN017 and B. lactis HN019 were examined bydetermining phagocyte (blood leukocytes and peritoneal macrophage)function, and quantifying concentrations of specific antibodies toprotein antigens used for mimicking responses to vaccines in mice.

[0037] The following experimental protocol was used:

[0038] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.

[0039] 2. Mice were randomly allocated to different treatment groups(Table 4)

[0040] 3. Mice were fed L. acidophilus HN017, L. rhamnosus HN001 or B.lactis HN019 (10⁹ cfu/day) in 50 μl skim milk for 10 days. Control micereceived 50 μl of skim milk powder only.

[0041] 4. All mice received skim milk powder based diet throughout theexperiment.

[0042] Blood leukocytes and macrophages from mice receiving L.acidophilus HN017, L. rhamnosus HN001 or B. lactis HN019 showedsignificantly greater phagocytic capacity compared with cells fromcontrol mice (Table 4). The production of oxygen radicals (oxidativeburst) by leukocytes from probiotic fed mice was also higher than thecontrol mice (data not shown). TABLE 4 The effect of dietary L.acidophilus HN017, L. rhamnosus HN001 and B. lactis HN019 on phagocytefunction in mice % Blood % Peritoneal leukocytes with macrophage withTreatment phagocytic activity phagocyte activity Control 14.33 ± 0.8766.1 ± 3.5 L. acidophilus HN017  22.7 ± 1.21** 79.0 ± 1.0** L. rhamnosusHN001 24.84 ± 0.93** 80.5 ± 1.8** B. lactis HN019 23.19 ± 0.95** 77.4 ±2.6*

[0043] The concentration of specific IgG antibodies in the sera and inthe intestinal washings of mice receiving L. acidophilus HN017, L.rhamnosus HN001 or B. lactis HN019 was also greater than those ofcontrol mice (Table 5). TABLE 5 The effect of dietary L. acidophilusHN017, L rhamnosus HN001 and B. lactis HN019 on serum and mucosalantibody responses Serum antibody Mucosal antibody response responseTreatment (units/ml) (units/ml) Control  80.2 ± 6.0 1350 ± 96.0 L.acidophilus HN017 134.6 ± 25.2* 1548 ± 270.0 L. rhamnosus HN001 118.5 ±12.5** 1512 ± 198.0 B. lactis HN019 158.1 ± 51.6*** 1548 ± 234.0 # mean± standard error. Significant differences (Students t test) fromcontrol:

EXAMPLE 4 Immunostimulating Effects Following Supplementation with LABfor Four Weeks

[0044] The immunostimulating effects of L. acidophilus HN017, L.rhamnosus HN001, and B. lactis HN019 were assessed in mice using thefollowing experimental protocol:

[0045] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.

[0046] 2. Mice were randomly allocated (18/group) to different treatmentgroups.

[0047] 3. After acclimatisation (for 7 days), mice were given 10⁹ cfu(per day) L. acidophilus HN017, L. rhamnosus HN001, or B. lactis HN019,in 50 μl skim milk, for 28 days (from day 0 to day 28). Control micereceived 50 μl skim milk (without any micro-organisms) only.

[0048] 4. Mice were offered a skim milk powder based-diet and water adlibitum, throughout the experiment.

[0049] 5. Immunostimulating effects were assessed by monitoringphagocytic activity of blood leukocytes and peritoneal macrophages,NK-cell activity of splenic lymphocytes, lymphocyte proliferation(spleen cells) responses to a T-cell mitogen, ConA (an indicator ofcell-mediated immunity) and antibody responses to Tetanus vaccine.

[0050] As seen in Table 6, leukocytes (neutrophils, monocytes andmacrophages) from mice receiving L. acidophilus HN017, L. rhamnosusHN001, or B. lactis HN019 exhibited significantly greater phagocyticactivity (an indicator of natural immunity) than leukocytes from controlmice. TABLE 6 The effect of dietary L. acidophilus HN017, L. rhamnosusHN001, and B. lactis HN019 in mice % Blood leukocytes with % Peritonealmacrophages Treatment phagocytic activity with phagocytic activityControl 15.5 72.67 L. acidophilus HN017 29.4** 82.2* L. rhamnosus HN00124.2** 82.8** B. lactis HN019 31.1** 83.0**

[0051] Consumption of L. acidophilus HN017, L. rhamnosus HN00 1, or B.lactis HN019 for 28 days also resulted in an increase in the NK-cellactivity, lymphocyte proliferation responses to ConA and antibodyresponses to Tetanus vaccine. For all these indicators ofimmunocompetence, mice receiving L. acidophilus HN017, L. rhamnosusHN001, or B. lactis HN019 had higher responses than those of controlmice (Table 7).

[0052] Together these results show that supplementation for extendedperiods with L. acidophilus HN017, L. rhamnosus HN001, or B. lactisHN019 is able to induce a sustained enhancement in several aspects ofnatural and acquired immunity. TABLE 7 The effect of dietary L.acidophilus HN017, L. rhamnosus HN001, and B. lactis HN019 on NK cellactivity and lymphocyte proliferation responses to ConA and antibodyresponses to Tetanus vaccine. Lymphocyte Antibody NK cell proliferationresponses to ConA activity to ConA Tetanus vaccine Treatment (%)(absorbance) (units/ml) Control 8.8  1.4 ± 0.125 402.5 ± 41.4  L.acidophilus HN017 9.9  1.6 ± 0.44  923.9 ± 116.0* L. rhamnosus HN00111.5  1.8 ± 0.1*  711.5 ± 127.2* B. lactis HN019 10.5  1.7 ± 0.5*  844.6± 134.7* #mice were immunised with Tetanus vaccine (50 μl/dose, CSL,Australia) on days 7 and 21. The concentration of specific antibodieswere determined using an ELISA; antigen supplied by the vaccinemanufacturers (CSL, Australia) was used for coating plates. Values areleast square means of 18 mice. Significant differences (the SASanalysis): *P < 0.05.

EXAMPLE 5 Enhancement of Natural and Acquired Immunity Using Fermentedversus Unfermented Products

[0053] The aim was to assess the immunoenhancing efficacy of yoghurtmade (fermented) using the probiotic strain L. rhamnosus HN001 comparedto unfermented product containing L. rhamnosus HN001. Theimmunoenhancing effects were examined by determining the phagocytefunction (peripheral blood leukocytes and peritoneal macrophages) andlymphocyte proliferative responses to a B-cell mitogen (LPS).

[0054] The following experimental protocol was used:

[0055] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.

[0056] 2. Mice were randomly allocated to different treatment groups.

[0057] 3. Control mice received a whole milk powder-based dietthroughout the experiment.

[0058] 4. Test mice received 2.5 g yoghurt made using L. rhamnosus HN001(10⁹ cfu/day) or 2.5 g whole milk containing L. rhamnosus HN001 (10⁹cfu/day) per day as well as a whole milk powder based diet for 14 days.

[0059] Results

[0060] Mice receiving yoghurt made with L. rhamnosus HN001 or whole milkcontaining L. rhamnosus HN001 displayed a significantly higher level ofphagocytic activity of peripheral blood leukocytes than was observed inmice receiving the control diet (FIG. 1). This increase was seenirrespective of whether the L. rhamnosus HN001 was delivered in theyoghurt (fermented with L. rhamnosus HN001) or unfermented productcontaining L. rhamnosus HN001. There was no difference in the level ofphagocytic activity between mice receiving the fermented yoghurt madeusing L. rhamnosus (HN001) compared to unfermented WMP productcontaining L. rhamnosus (HN001).

[0061] Both the unfermented and L. rhamnosus HN001 fermented product fedmice showed higher lymphocyte proliferative responses to LPS than thecontrol mice (Table 8). There was no significant difference in theresponse between mice receiving unfermented product containing L.rhamnosus HN001 and mice receiving product fermented with L. rhamnosusHN001. TABLE 8 The effect of fermented and unfermented L. rhamnosusHN001 on lymphocyte proliferative responses in mice Lymphocyteproliferation to Treatment LPS (absorbance) Control (WMP) 0.4699 ± 0.028WMP Fermented with L. rhamnosus HN001 0.5361 ± 0.028 Unfermented WMPwith L. rhamnosus 0.5518 ± 0.028* HN001 # 16 hrs of the 96 hrincubation. Values are means ± standard error. Significant differences(Students t test) from the control: *P = 0.05.

[0062] Together these results suggest that supplementation with L.rhamnosus HN001 enhances a range of immune functions includingphagocytic activity and lymphocyte cell proliferation. L. rhamnosusHN001 presented in either fermented or unfermented product is effectiveat eliciting enhancement of immune function, with fermented productgiving a greater response for some functions and unfermented beingsuperior in others.

EXAMPLE 6 Enhancement of Natural and Acquired Immunity by L. rhamnosusHN067

[0063] Experiment 1

[0064] The immunoenhancing effects of L. rhamnosus HN067 were examinedby monitoring phagocytic capacity of peripheral blood leukocytes andperitoneal macrophages (indicator of non-specific immunity), andquantifying concentrations of specific antibodies to an immunisationantigen, cholera toxin (used for mimicking responses to entericvaccines) in mice.

[0065] The following experimental protocol was used:

[0066] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.They were fed on a skim milk-based diet throughout the experiment.

[0067] 2. Mice in the test group (n=6) were orally administered L.rhamnosus HN067 (10⁹ cfu/day) in 50 μl skim milk for 10 days. Controlmice (n=6) received 50 μl of skim milk powder (without any LAB) only.

[0068] Results

[0069] Blood leukocytes and peritoneal macrophages from mice receivingL. rhamnosus HN067 showed significantly greater phagocytic activity(enhanced phagocyte function) compared with cells from control mice. Theresults are set out in Table 9 below. TABLE 9 The effect of dietary L.rhamnosus HN067 on phagocyte function % Peritoneal % Blood leukocyteswith macrophages with Treatment phagocytic activity phagocytic activityControl 13.1 ± 1.5 76.4 ± 1.9 L. rhamnosus HN067 23.7 ± 1.5** 87.2 ±1.9* # (the SAS program) from the control: *P = 0.0005, **P = 0.0001.

[0070] The concentration of specific antibodies to cholera toxin, anantigen used for oral immunisation, in the sera and in the intestinalwashings of mice receiving L. rhamnosus HN067 was also significantlygreater than those of control mice (Table 10). TABLE 10 The effect ofdietary supplementation with L. rhamnosus HN067 on serum and mucosalantibody responses to cholera toxin Serum Mucosal antibody anitbodyresponse response Treatment (units/ml) (units/ml) Control 63.1 ± 43.21969.7 ± 279.5 L. rhamnosus HN067  246.5 ± 43.2**  2995.5 ± 465.2*

[0071] Experiment 2

[0072] The immunostimulating effects of L. rhamnosus HN067 were assessedin mice using the following experimental protocol:

[0073] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.They were offered skim milk powder based diet and water ad libitum,throughout the experiment.

[0074] 2. After acclimatisation for 7 days, mice in group 1 (n=20) wereorally administered with 10⁹ cfu (per day) L. rhamnosus (HN067) in 50 μlskim milk (group 1 n=20) for 14 days. Control nice (group 2, n=20)received skim milk without any microorganisms.

[0075] 3. Immunostimulating effects were assessed by monitoringphagocytic activity of blood leukocytes and peritoneal macrophages, andspleen lymphocyte proliferation responses to phytohaemagglutinin (PHA)and lipopolysaccharide (LPS) (T and B-cell mitogens respectively).

[0076] Results

[0077] Blood leukocytes and peritoneal macrophages from mice receivingL. rhamnosus HN067 exhibited significantly greater phagocytic activity(an indicator of natural immunity) than leukocytes and macrophages fromcontrol mice (Table 11). TABLE 11 The effect of dietary L. rhamnosusHN067 on phagocyte function in mice % Blood % Peritoneal Leukocytes withmacrophages Treatment phagocytic activity with phagocytic activityControl 13.7 ± 0.07 64.6 ± 2.1 L. rhamnosus HN067 22.5 ± 0.07** 75.8 ±1.7*

[0078] Mice receiving L. rhamnosus HN067 for 14 days also displayedhigher lymphocyte proliferation responses to PHA and LPS compared withcontrol mice (Table 12). TABLE 12 The effect of L. rhamnosus HN067supplementation on lymphocyte proliferation responses to PHA and LPSConA Lymphocyte Lymphocyte Treatment proliferation to PHA proliferationto LPS Control 1.18 ± 0.08 0.99 ± 0.07 L. rhamnosus HN067 1.37 ± 0.07*1.24 ± 0.06**

[0079] In summary, mice receiving L. rhamnosus HN067 displayedsignificant enhancement of a range of host immune responses includingleukocyte phagocytic function, antibody responses to oral immunisation,and lymphocyte proliferation responses to T and B-cell mitogens. Bloodleukocytes (neutrophils and monocytes) and macrophages are majoreffectors of natural immunity and play a major role in protectionagainst microbial infections. A correlation between in vitro lymphocyteproliferation responses to mitogens (T- and B-cell mitogens) andimmunocompetence of an individual is also well documented. Therefore,these results suggest that supplementation with L. rhamnosus HN067 isable to enhance several aspects of natural and acquired immunity.

EXAMPLE 7 Enhancement of Natural and Acquired Immunity Using Live andHeat Killed L. rhamnosus HN001

[0080] The aim of the present study was to investigate theimmunoenhancing effects of the probiotic strain L. rhamnosus HN001 whenpresented in either the live or heat killed form. The effect on immunefunction was assessed by determining phagocytic activity of peripheralblood leukocytes. The effect of live and heat killed L. rhamnosus HN001on humoral immunity was investigated by immunising mice with choleratoxin, and measuring the concentrations of specific antibodies produced.

[0081] The following experimental protocol was used:

[0082] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.

[0083] 2. Mice were randomly allocated to different treatment groups.

[0084] 3. Control mice received a skim milk powder based diet throughoutthe experiment.

[0085] 4. Test mice receive either 10⁹ cfu/day of live L. rhamnosusHN001 or to cfu/day heat killed L. rhamnosus HN001 per day as well as askim milk powder-based diet for 14 days.

[0086] 5. Mice were orally immunised with cholera toxin on day 0 and day7 of feeding.

[0087] Results

[0088]L. rhamnosus HN001 feeding significantly enhanced the level ofphagocytic activity of peripheral blood leukocytes compared to micereceiving the control diet (FIG. 2). This increase was seen irrespectiveof whether the L. rhamnosus HN001 was delivered in the live or heatkilled form. There was no difference in the level of phagocytic activitybetween the mice receiving live L. rhamnosus HN001 compared to heatkilled L. rhamnosus HN001.

[0089] Feeding of both live and dead L. rhamnosus HN001 induced anincrease in both serum and mucosal antibody responses compared to thecontrol mice. However, the level of response was significantly greaterin the mice fed the live L. rhamnosus HN001 (Table 13). TABLE 13 Theeffect of live and heat killed L. rhamnosus HN001 on serum and mucosalantibody responses to Cholera Toxin in mice Serum antibody Mucosalantibody Treatment response (units/ml) response (units/ml) Control 88.69 ± 18.52  708.6 ± 146.9 Live L. rhamnosus HN001 214.89 ± 62.33*2054.5 ± 285.8*** Heat Killed L. rhamnosus 174.89 ± 44.78 1533.6 ± 319.3HN001

[0090] These results suggest that both live and heat killed L. rhamnosusHN001 are able to enhance aspects of natural and acquired immunity inmice.

EXAMPLE 8 Anti-infection Properties of B. lactis HN019 and L. rhamnosusHN001

[0091] The aims of the current study were to:

[0092] 1. Assess the protection efficacy of B. lactis HN019 and L.rhamnosus HN001 against the gastrointestinal pathogen Salmonellatyphimurium.

[0093] 2. Determine the role of immunostimulation induced by B. lactisHN019 and L. rhamnosus HN001 in protection against S. typhimuriuminfection in mice.

[0094] Anti-infection properties were assessed by measurement ofbacterial translocation to the liver and spleen. The immunoenhancingeffects were examined by determining the phagocyte function (peripheralblood leukocytes and peritoneal macrophages) and lymphocyteproliferative responses to a T-cell mitogen (PHA).

[0095] The following experimental protocol was used:

[0096] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were used.

[0097] 2. Mice were randomly allocated to 4 difference treatment groupsand were individually housed.

[0098] 3. All mice received a skim milk powder based diet throughout theexperiment

[0099] 4. Test mice commenced daily feeding of B. lactis HN019 or L.rhamnosus HN001 (10⁹ cfu/day) 7 days prior to challenge, and continuedfor the duration of the trial.

[0100] 5. Mice administered with B. lactis HN019 or L. rhamnosus HN001and a control group (no LAB) were orally challenged with Salmonellatyphimurium (ATCC 1772) 8×10⁵ cfu/day for 5 days starting on day 7.

[0101] 6. An uninfected control group did not receive S. typhimuriumchallenge.

[0102] 7. On day 6 after challenge mice were used for the measurement ofbacterial translocation to the liver and spleen, and for immune functionassessment.

[0103] Results

[0104] Both the B. lactis HN019 and L. rhamnosus HN001 supplemented miceshowed significantly lower levels of bacterial translocation into theliver and spleen than the S. typhimurium alone fed mice (FIG. 3).

[0105] Challenge infection resulted in a significant suppression ofphagocyte function (FIG. 4); the phagocytic activity of control micechallenged with S. typhimurium was significantly lower than that of theuninfected mice. However, infection with S. typhimurium had no effect onthe phagocytic ability of peripheral blood leukocytes of micesupplemented with B. lactis HN019 or L. rhamnosus HN001. This was shownby similar levels of phagocytic activity in mice supplemented with B.lactis HN019 or L. rhamnosus HN001 and challenged with S. typhimuriumand the normal uninfected control mice.

[0106] Both the B. lactis HN019 and L. rhamnosus HN001 supplemented miceshowed higher lymphocyte proliferative responses to PHA than the S.typhimurium challenged control (FIG. 5). There was no significantdifference in the response between mice receiving B. lactis HN019 or L.rhamnosus HN001 and the uninfected control mice.

[0107] Together these results suggest that supplementation with B.lactis HN019 or L. rhamnosus HN001 is able to confer protection againstenteric pathogens such as Salmonella typhimurium. Enhanced resistance toinfection is accompanied by an increase in immune performance.

1. A biologically pure culture of either L. rhamnosus HN001 AGAL depositnumber NM97/09514 dated Aug. 18, 1997 or L. rhamnosus HN067 AGAL depositnumber NM97/01925 dated Feb. 11,
 1998. 2. A biologically pure culture ofL. rhamnosus HN001 AGAL deposit number NM97/09514 dated Aug. 18, 1997.3. A biologically pure culture of L. rhamnosus HN067 AGAL deposit numberNM97/01925.
 4. A composition of a biologically pure culture of any oneof L. rhamnosus HN001 as claimed in claim 2, L. rhamnosus HN067 asclaimed in claim 3, B. lactis HN019 AGAL deposit number NM97/09513 datedAug. 18, 1997 or L. acidophilus HN017 AGAL deposit number NM97/09515dated Aug. 18, 1997 in an immunostimulating concentration, with aphysiologically acceptable excipient or diluent.
 5. A composition asclaimed in claim 4 containing any two or more of said strains.
 6. Acomposition as claimed in claim 4 or 5 wherein said physiologicallyacceptable excipient or diluent is a food.
 7. A composition as claimedin claim 6 wherein said food is any one of cultured milk, yoghurt,cheese, milk drink or milk powder.
 8. A composition as claimed in claim4 or 5 which is a pharmaceutical composition and wherein said excipientor diluent is pharmacologically acceptable excipient or diluent. 9.Immunity enhancing physiologically acceptable biologically pure strainsof homologues or mutants of any one of the strains: L. acidophilusHN017, L. rhamnosus HN001, B. lactis HN019, or L. rhamnosus HN067.
 10. Amethod of enhancing natural and acquired immunity which comprisesadministering to a mammal a biologically pure cultures as claimed in anyone of claims 1-3 and 9 at an immunostimulating dosage rate.
 11. Amethod as claimed in claim 10 wherein biologically pure cultures of twoor three of the above-defined strains are present.
 12. A method ofenhancing natural and acquired immunity which comprises administering toa mammal a composition as claimed in any one of claims 4 to
 8. 13. Amethod as claimed in claim 10 wherein said physiologically acceptableexcipient or diluent is a food.
 14. A method as claimed in claim 10wherein said food is cultured milk, yoghurt, cheese, milk drink or milkpowder.